Heidmann_1983_Biochemistry_22_5452

Excitable acetylcholine receptor rich membrane fragments from Torpedo marmorata have been used to measure, in parallel, (1) the permeability response to the fluorescent cholinergic agonist Dns-C6-Cho (in the 0.1 microM to millimolar concentration range) characterized by both the initial rate of Li+ transport and the rate of channel closure using the rapid-mixing quench-flow technique and (2) the kinetics of interaction of Dns-C6-Cho with the acetylcholine receptor sites using the rapid-mixing stopped-flow technique. Analysis of the kinetics of Dns-C6-Cho binding in the millisecond to minute time scale leads to the identification of at least three conformational states of the acetylcholine receptor: a "low-affinity" one (approximately 50 microM) that can be interconverted in the fraction of a second to a transient state of "intermediate affinity" (approximately 1 microM), followed by the final stabilization, in the second to minute time range, of a state of "high affinity" (approximately 3 nM). Comparison of Dns-C6-Cho binding data with the permeability response to the same agonist demonstrates that the binding to the low-affinity conformation(s) of the acetylcholine receptor sites coincides with the triggering of the permeability increase--or "activation"--and the transitions to the intermediate- and high-affinity states with the two-step process of channel closing--or "desensitization". The data are interpreted in terms of a minimum four-state "allosteric" model for the acetylcholine receptor.